Overview: This research program addresses basic molecular and physiological processes of nociceptive (pain-sensing) transmission in the peripheral and central nervous systems (CNS) and new ways to effectively control pain. The molecular research is performed using animal and in vitro cell-based models. We concentrate on primary afferent pain-sensing neurons located in dorsal root ganglion (DRG) that send nerve fibers to skin and deep tissues and make connections within dorsal spinal cord, which is the first CNS site of synaptic information processing for pain. The mechanisms of transduction of physical pain stimuli are investigated through models of pathophysiological damage or using reductionist preparations such as primary DRG cultures or heterologous expression systems of ion channels or receptors. Our goals are (a) to understand the molecular and cell biological mechanisms of acute and chronic pain at the initial steps in the pain pathway, (b) to investigate mechanisms underlying human chronic pain disorders, (c) to explore neuronal plasticity and altered gene expression in persistent pain states, and (d) to use this knowledge to devise new treatments for pain. New Treatments for Pain: We address the new treatment goal through translational research coupled with human clinical trials to develop and introduce new molecular interventions for severe pain. Studies with the TRPV1 agonist resiniferatoxin (RTX) have resulted in a Phase I clinical trial for in patients with intractable pain from advanced cancer. RTX activates TRPV1, which is an inflammation-, heat-, and capsaicin-sensitive calcium/sodium ion channel that normally converts painful heat or acidic pH into nerve action potentials by opening the pore of the ion channel. The influx of ions depolarizes pain-sensing nerve endings and sends electrical signals to the spinal cord (which in turn sends the signals to the brain where we perceive pain). After binding to TRPV1, RTX props open the ion channel, causing intracellular calcium cytotoxicity. Depending on the route of administration this disables or deletes TRPV1 pain-sensing neurons, their nerve endings or axons (i.e., the nerve fiber). RTX produces very effective pain control in pre-clinical models. The central route involves administration into the cerebrospinal fluid around the spinal cord (intrathecal). After approval of the clinical protocol by our IRB and the Investigational New Drug application by the FDA, we have treated 12 patients with pain from advanced cancer. This study is nearly complete. We also published a study of injections around or directly into sensory ganglia, and, based on this approach, we will commence a new protocol for more localized cancer pain problems. Peripheral routes of RTX administration include direct injection into skin, joints, nerve bundles, or topically. Analgesia by these routes is long-lasting but temporary since nerve endings and axons regrow. Peripheral administration formed the basis of three reports, one in which we successfully treated experimental burn pain, another in which we successfully used RTX as a preemptive analgesic for surgical incision pain and a third report (in progress) in which we successfully treated clinical osteoarthritis (OA) pain by intraarticular injection in client owned dogs. The canine results demonstrated strong efficacy and a long duration of action (4 to >12 months) upon RTX injection into knee joints and strongly reinforce the objective of therapeutic use of vanilloid agonists for pain control and the translation to human patients. Early Translational Investigations: In collaboration, we have also examined the pharmacological activity of polyunsaturated ethanolamines and linoleic acid metabolites. These putative endovanilloids. could function as endogenous orthosteric agonists at TRPV1. In this cycle, we evaluated tissue biosynthetic pathways for new endogenous lipids. We published our discovery of two previously unknown lipids. One sensitized nerves to nociceptive stimuli, the other caused itch and, in humans, were associated with headache and itch conditions, respectively. In ongoing allied studies, we examined small chemicals that act as positive allosteric modulators (PAMs) of TRPV1 activation. By high throughput screening we discovered several new chemical compounds that enhance the activation of TRPV1 upon orthosteric agonist (capsaicin) binding or by elevated H+ ions. Medicinal chemistry efforts yielded DPM-32 and DPM-74, which are active in in vivo or in vitro assays. These experiments reveal a new approach to pain modulation and pain control and support the idea that TRPV1 agonist activity, induced in several different ways, has the potential to yield novel, non-narcotic, non-addictive, selective, long-lasting analgesic agents that may be effective in acute, persistent, or chronic pain disorders. During this cycle we also published a report on a protein therapeutic agent that is a conjugate between Substance P and a bioengineered Pseudomonas exotoxin. This agent is endocytosed by the substance P receptor expressing second order spinal cord dorsal horn neurons and the exotoxin moiety stops protein synthesis thereby killing the neuron and interrupting the pain pathway to the brain. This is a potent analgesic agent. We intend to test it in certain pain indications, including cancer pain and spinal cord injury pain. Molecular manipulations aimed at generating high expressing constructs are in progress. Basic Pain Mechanisms: Underlying the translational and clinical studies are investigations of molecular biology, neuronal function, behavior, and mechanisms of pain transduction. We are systematically investigating molecular alterations at the first three steps in the pain pathway beginning with injured peripheral tissue, the dorsal root ganglion and the dorsal (sensory) spinal cord in order to obtain a foundational, comprehensive, quantitative molecular understanding of nociceptive processes related to inflammation and nerve injury. Our studies reveal a complex, dynamic modulation of gene expression at all three steps. We are using a method called RNA-seq to sequence all of the mRNAs in a given tissue or cell population and have identified prominent roles for new, key molecules with distinct combinatorial patterns of expression among the three tissues. We also combine RNA-seq with genetically or pharmacologically manipulated mice and rats and in humans with copy number variants that affect pain sensitivity, one being a hemideletion on chromosome 11 and the other a duplication on chromosome 7. The results are both compelling and informative, and define previously unidentified genetic components governing pain sensitivity. We also have used RNA-seq to define genes involved in inherited peripheral neuropathies and pain channelopathies. These investigations provide a much better quantitative assessment of genes expressed in tissues and neurons of the nociceptive circuit in basal conditions, after pathophysiological manipulations, and in human genetic variations that affect pain sensitivity. Through this basic research we aim to obtain a deeper understanding of mechanisms that trigger acute pain and sustain chronic pain and to identify molecular components to control pain.